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Published on: October 21, 2012
Apoptosis assays with lymphoma cell lines: problems and pitfalls.
1Garden State Cancer Center at the Center for Molecular Medicine and Immunology, 520 Belleville Avenue, Belleville, NJ 07109, USA. mjmattes@gscancer.org
British Journal of Cancer
|March 8, 2007
Summary
Distinguishing apoptosis from other cell death types is crucial. Many common apoptosis assays, including DNA content analysis and mitochondrial membrane potential assays, can yield inaccurate results, complicating cell death determination.
Area of Science:
- Cell biology
- Cancer research
- Biochemistry
Background:
- Determining the precise mechanism of tumor cell death following cytotoxic treatment is essential for effective cancer therapy.
- Various assays are employed to differentiate between apoptosis and other forms of cell death, but their accuracy is under scrutiny.
Purpose of the Study:
- To critically evaluate the reliability of commonly used assays for detecting apoptosis.
- To identify potential pitfalls and confounding factors in current methods for assessing cell death.
Main Methods:
- Analysis of sub-G1 DNA content using propidium iodide staining.
- Assessment of mitochondrial membrane potential.
- Evaluation of annexin V staining.
- Investigation of non-specific antibody binding in irradiated cells.
- Consideration of cell aggregation in antibody crosslinking experiments.
Main Results:
- Sub-G1 DNA content and loss of mitochondrial membrane potential do not reliably distinguish apoptosis from necrosis without additional controls.
- Cellular fragments can interfere with annexin V staining, complicating size threshold determination.
- Increased non-specific antibody binding in irradiated cells can lead to misinterpretation of antigen expression.
- Cell aggregation can introduce artifacts and mechanical stress, impacting cell death assays.
Conclusions:
- Widely used apoptosis assays may not accurately distinguish between apoptosis and other cell death modalities.
- Careful consideration of assay limitations, including potential artifacts and the need for appropriate controls, is necessary for accurate determination of cell death mechanisms.
